Beta catenin nucleic acid inhibitor molecule

ABSTRACT

Provided herein is a potent, optimized β-catenin nucleic acid inhibitor molecule with a unique pattern of modified nucleotides. Also provided are methods and compositions for reducing β-catenin expression and methods and compositions for treating cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/573,999, entitled “BETA CATENIN NUCLEIC ACID INHIBITOR MOLECULE,”filed on Oct. 18, 2017, and U.S. Provisional Patent Application No.62/614,206, entitled “REDUCING BETA-CATENIN AND IDO EXPRESSION TOPOTENTIATE IMMUNOTHERAPY,” filed on Jan. 5, 2018, the entire contents ofwhich are incorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Oct. 11, 2018, is named0243_0008-PCT_SL.txt and is 19,219 bytes in size.

BACKGROUND

β-catenin, an oncogene, is a key mediator of Wnt signaling in cells.β-catenin serves several cellular functions at multiple cellularlocations, including the plasma membrane, where β-catenin contributes tothe stabilization of intercellular adhesive complexes, the cytoplasmwhere β-catenin levels are regulated, and the nucleus where β-catenin isinvolved in transcriptional regulation and chromatin interactions.

Mutations in β-catenin (encoded by the CTNNB1 gene in humans) have beenspecifically associated with colorectal, desmoid, endometrial, gastric,hepatocellular, hepatoblastoma, kidney (Wilms' tumor), medulloblastoma,melanoma, ovarian (endometrioid), pancreatic, pilomatricoma, prostate,thyroid (anaplastic) and uterine (endometrium) cancers (Polakis P. GenesDev. 14: 1837-51; Samowitz et al. Cancer Res. 59: 1442-4; Iwao et al.Cancer Res. 58: 1021-6; Mirabelli-Primdahl et al. Cancer Res. 59:3346-51; Shitoh et al. J Clin Path. 52: 695-6; Tejpar et al. Oncogene18: 6615-20; Kitaeva et al. Cancer Res. 57: 4478-81; Sparks et al.Cancer Res. 58: 1130-4; Miyaki et al. Cancer Res. 59: 4506-9; Park etal. Cancer Res. 59: 4257-60; Huang et al. Am J Pathol. 155: 1795-801;Nhieu et al. Am J Pathol. 155: 703-10; Legoix et al. Oncogene 18:4044-6; Jeng et al. Cancer Lett. 152: 45-51; Koch et al. Cancer Res. 59:269-73; Wei et al. Oncogene 19: 498-504; Koesters et al. Cancer Res. 59:3880-2; Maiti et al. Cancer Res. 60: 6288-92; Zurawel et al. Cancer Res.58: 896-9; Gamallo et al. Am J Pathol. 155: 527-36; Palacios and GamalloCancer Res. 58: 1344-7; Wright et al. Int J Cancer 82: 625-9; Gerdes etal. Digestion 60: 544-8; Chan et al. Nat Genet. 21: 410-3; Voeller etal. Cancer Res. 58: 2520-3; Garcia-Rostan et al. Cancer Res. 59: 1811-5;Fukuchi et al. Cancer Res. 58: 3526-8).

The β-catenin/Wnt pathway is consistently activated in over 80% ofcolorectal cancers. The β-catenin/Wnt pathway is also consistentlyactivated in over 50% of hepatocellular carcinoma (HCC) patients.Activated Wnt signaling and nuclear β-catenin correlate with recurrenceof disease and poor prognosis (Takigawa et al. 2008, Curr Drug TargetsNovember; 9 (11):1013-24).

Mutations in the β-catenin gene include truncations that lead todeletion of part of the N-terminus of β-catenin or point mutations thataffect the serine and threonine residues that are targeted by componentsof the cytoplasmic destruction complex, such as GSK3α/β or CKIα, thatmediate the phosphorylation of β-catenin and target its degradation bythe proteosome. These mutant β-catenin proteins are refractory tophosphorylation and thus escape proteasomal degradations. Consequently,β-catenin accumulates within affected cells. Stabilized andnuclear-localized β-catenin is a hallmark of nearly all cases of coloncancer. (Clevers, H., 2006, Cell 127:469-480). Morin et al. demonstratedthat mutations of β-catenin that altered phosphorylation sites renderedthe cells insensitive to APC-mediated down-regulation of β-catenin andthat this disrupted mechanism was important to colorectal tumorigenesis.(Morin et al., 1997, Science 275:1787-1790).

Despite advances in understanding how β-catenin functions as a keymediator of Wnt signaling in cells and how mutations and/or alteredexpression of β-catenin can play a role in tumorigenesis, there remainsa need for compositions that can be used to treat disease associatedwith CTNNB1 expression, such as cancer.

SUMMARY

This application provides a potent, optimized double-stranded β-cateninnucleic acid inhibitor molecule having a unique pattern of modifiednucleotides, including a unique pattern of 2′-Fluoro (“2′-F) and2′-O-methyl (“2′-OMe” or “2′-OCH₃”) modifications at the 2′-carbon ofthe sugar moiety of the majority of nucleotides in the molecule. Withoutintending to be bound by any theory, it appears that the unique patternof nucleotide modifications provides improved properties to theβ-catenin nucleic acid inhibitor molecule, such as one or more ofreduced immunogenicity, improved reduction of β-catenin mRNA expression,improved Ago2 binding, or improved inhibition of tumor growth.

In one embodiment, the optimized β-catenin nucleic acid inhibitormolecule comprises a sense (or passenger) strand comprising orconsisting of the nucleic acid of SEQ ID NO: 11 and an antisense (orguide) strand comprising or consisting of the nucleic acid of SEQ ID NO:12, and a region of complementarity between the sense strand and theantisense strand of 26 nucleotides. The antisense strand includes 2single-stranded nucleotides at its 3′ terminus and 10 single-strandednucleotides at its 5′ terminus. In certain embodiments, the sense strandconsists of the nucleic acid of SEQ ID NO: 11 and the antisense strandconsists of the nucleic acid of SEQ ID NO: 12.

In one embodiment, the optimized β-catenin nucleic acid inhibitormolecule is a double-stranded nucleic acid inhibitor molecule comprisinga sense strand and an antisense strand;

wherein the sense strand comprises or consists of the nucleic acidsequence of SEQ ID NO: 13 and the antisense strand comprises or consistsof the nucleic acid sequence of SEQ ID NO: 14 and the sense strand andantisense strand together form a duplex region of 26 base pairs and theantisense strand includes 2 single-stranded nucleotides at its 3′terminus and 10 single-stranded nucleotides at its 5′ terminus;

wherein the sugar moiety of each of nucleotides 1, 3, 7, 9-11, 13, 15,19, and 20 of SEQ ID NO: 13 is modified with a 2′-F, the sugar moiety ofeach of nucleotides 2, 4-6, 8, 12, 14, 16-18, and 24 of SEQ ID NO: 13 ismodified with a 2′-OCH₃, nucleotides 25 and 26 of SEQ ID NO: 13 aredeoxyribonucleotides, and nucleotides 21-23 of SEQ ID NO: 13 areribonucleotides; and

wherein the sugar moiety of each of nucleotides 2, 6, 19, 21, 23, 25,30, 31, 33, and 35 of SEQ ID NO: 14 is modified with a 2′-F, the sugarmoiety of each of nucleotides 1, 3-5, 7-9, 11-15, 20, 22, 24, 26, 32,34, and 36-38 of SEQ ID NO: 14 is modified with a 2′-OCH₃, nucleotide 10of SEQ ID NO: 14 is a deoxyribonucleotide, and nucleotides 16-18 and27-29 of SEQ ID NO: 14 are ribonucleotides.

One aspect is directed to a method of treating cancer in a subject,comprising administering to the subject a therapeutically effectiveamount of the optimized β-catenin nucleic acid inhibitor molecule, asdescribed herein. In certain embodiments, the method further comprisesadministering a therapeutically effective amount of an immunotherapeuticagent. In certain embodiments, the subject is a human.

Another aspect is directed to a pharmaceutical composition comprisingthe optimized β-catenin nucleic acid inhibitor molecule, as describedherein. In certain embodiments, the pharmaceutical compositioncomprising the optimized β-catenin nucleic acid inhibitor molecule isfor use in treating cancer. In certain embodiments, the pharmaceuticalcomposition is administered in combination with an immunotherapeuticagent, such as an anti-CTLA-4 monoclonal antibody, an anti-PD-1monoclonal antibody, an anti-PD-L1 monoclonal antibody, or a combinationof an anti-CTLA-4 monoclonal antibody and an anti-PD-1 monoclonalantibody.

In certain embodiments of the method or the pharmaceutical composition,the cancer is a non-Wnt activated cancer. In other embodiments, thecancer is a Wnt activated cancer. In certain embodiments of the methodor the pharmaceutical composition, the non-Wnt activated cancer is amelanoma, a neuroblastoma, or a renal cancer.

In certain embodiments of the method or composition, the optimizedβ-catenin nucleic acid inhibitor molecule, as described herein, isformulated with a lipid nanoparticle. In certain embodiments, the lipidnanoparticle comprises core lipids and envelope lipids, wherein the corelipids comprise a first cationic lipid and a first pegylated lipid andwherein the envelope lipids comprise a second cationic lipid, a neutrallipid, a sterol, and a second pegylated lipid. In certain embodiments,the first cationic lipid is DL-048, the first pegylated lipid isDSG-MPEG, the second cationic lipid is DL-103, the neutral lipid isDSPC, the sterol is cholesterol, and the second pegylated lipid isDSPE-MPEG.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate certain embodiments, and togetherwith the written description, serve to explain certain principles of thecompositions and methods disclosed herein.

FIG. 1A schematically shows the structures of three, exemplarynon-extended double-stranded nucleic acid inhibitor molecules thattarget the β-catenin gene and have different nucleotide modificationpatterns: NonExtended M1 (NEX M1); NonExtended M2 (NEX M2); andNonExtended M3 (NEX M3). In this schematic, unshaded nucleotidesrepresent ribonucleotides, striped nucleotides contain 2′-OMemodifications, shaded bases contain 2′-F modifications, and nucleotidesoutlined with black boxes represent deoxyribonucleotides. The passengerand guide strands of NEX M1 correspond to SEQ ID NO: 1 and SEQ ID NO: 2,respectively. The passenger and guide strands of NEX M2 correspond toSEQ ID NO: 3 and SEQ ID NO: 4, respectively. The passenger and guidestrands of NEX M3 correspond to SEQ ID NO: 5 and SEQ ID NO: 6,respectively. Also shown are the IC50 values (pM) for the threemolecules.

FIG. 1B shows the interferon response (as measured by IFIT1 induction)of different non-extended double-stranded nucleic acid inhibitormolecules in human peripheral blood mononuclear cells (PBMCs). NEX M1induced significant elevation of IF1T1 while NEX M2 showed moderateelevation. NEX M3, on the other hand, showed only minimal elevation ofIFIT1.

FIG. 2A schematically shows the structures of three, exemplary extendeddouble-stranded nucleic acid inhibitor molecules that target thebeta-catenin gene and have different nucleotide modification patterns:Extended M1 (EX M1); Extended M2 (EX M2); and Extended M3 (EX M3). Inthis schematic, unshaded nucleotides represent ribonucleic acid bases,striped nucleotides represent 2′-OMe modified bases, shaded basesrepresent 2′-F modified bases, and nucleotides outlined with black boxesrepresent deoxyribonucleic acid bases. The passenger and guide strandsof EX M1 correspond to SEQ ID NO: 7 and SEQ ID NO: 8, respectively. Thepassenger and guide strands of EX M2 correspond to SEQ ID NO: 9 and SEQID NO: 10, respectively. The passenger and guide strands of EX M3correspond to SEQ ID NO: 11 and SEQ ID NO: 12, respectively. Also shownare the IC50 values (pM) for the three molecules.

FIG. 2B shows the interferon response (as measured by IFIT1 induction)of different extended double-stranded nucleic acid inhibitor moleculesin human peripheral blood mononuclear cells (PBMCs). EX M1 inducedsignificant elevation of IF1T1 while EX M2 showed moderate elevation.NEX M3, on the other hand, showed essentially no elevation of IFIT1.

FIG. 3A shows the results from an assay in which relative immunogenicitywas assessed by measuring the impact on interferon levels for NEX M1,NEX M3, and EX M3 when mixed with human PBMCs from four differentdonors. NEX M3 and EX M3 showed little or no interferon response for allfour donors, whereas NEX M1 showed a modest interferon response forDonors 1, 2, and 4 and a substantial interferon response for Donor 3(hypersensitive).

FIG. 3B shows the amount of beta-catenin mRNA knockdown (“potency”) inLS411N tumors at different time points (24 and 120 hours post-treatment)obtained from mice treated with NEX M1, NEX M3, and EX M3. At 24 hours,EX M3 demonstrated slightly better potency when compared to EX M2 andNEX M3 and similar potency compared to NEX M1. At 120 hours aftertreatment, EX M3 showed an improved potency as compared to NEX M1.

FIGS. 4A-D show graphs or images of results from various assayscomparing the NEX M1 and EX M3 constructs.

FIG. 4A shows that in mice bearing LS411N tumors and treated with NEX M1and EX M3, EX M3 was present at much higher levels in tumor tissue atday 3 after dosing.

FIG. 4B shows that in mice bearing LS411N tumors and treated with NEX M1and EX M3, EX M3 exhibited increased Argonaute (Ago) 2binding/RNA-induced silencing complex (RISC) loading efficiencies intumor tissue at day 3 after dosing.

FIG. 4C shows that in mice bearing SW403 tumors and treated with NEX M1and EX M3, NEX M1 inhibited tumor growth by about 55% relative tovehicle-treated animals, whereas EX M3 inhibited tumor growth by over80%. The x-axis lists the days post-implant (of SW403 tumors) with thearrows indicating the days on which NEX M1, EX M3, or control wasadministered (at a dosage of 3 mg/kg).

FIG. 4D shows by immunohistochemistry that there was a substantialdecrease in beta-catenin protein levels in SW403 tumors obtained at theend of the study depicted in FIG. 4C.

FIG. 5 shows one non-limiting embodiment of a lipid nanoparticle (LNP)that can be used to formulate the β-catenin nucleic acid inhibitormolecule. The LNP includes the following core lipids: DL-048 (cationiclipid) and DSG-MPEG (pegylated lipid), and the following envelopelipids: DL-103 (cationic lipid), DSPC, cholesterol, and DSPE-MPEG(pegylated lipid).

FIG. 6 shows a simplified diagram of the Wnt signaling pathway. The leftside depicts a cell where the Wnt ligand is not bound to its surfacereceptor, β-catenin is sequestered in a destruction complex and targetedfor ubiquitination and degradation, and target genes are repressed. Theright side depicts a cell after the Wnt ligand has bound its surfacereceptor, where the destruction complex disassembles, stabilizedβ-catenin is released and travels to the nucleus, and target genes areactivated.

FIG. 7A shows the treatment schedule for Balb/C mice that were implantedwith Wnt-activated, 4T1 tumors and treated with placebo or EX M3, asdescribed in Example 5.

FIG. 7B shows by immunohistochemistry that EX M3 treatment decreasesβ-catenin levels and increases CD8 T-cell infiltration but does notsignificantly reduce IDO1 levels in 4T1 tumors.

FIG. 7C shows that two cycles of EX M3 treatment inhibits tumor growthas compared to placebo in 4T1 tumors that were implanted into Balb/Cmice.

FIG. 8A shows the treatment schedule for Balb/C mice that were implantedwith 4T1 tumors and treated with PBS or EX M3, as described in Example5.

FIG. 8B shows by flow cytometry analysis that EX M3 treatment of 4T1tumors increases CD8+ T cells, increases multiple checkpoint molecules(PD-1, LAG-3+, and Tim-3+), and increases regulator T cells (Tregs) butdoes not significantly alter the number of myeloid derived suppressorcells (MDSC) in the tumor m icroenvironment.

FIG. 9A shows the treatment schedule for Balb/C mice that were implantedwith 4T1 tumors and treated with vehicle or an IDO inhibitor (IDOi)called epacadostat, as described in Example 6.

FIG. 9B shows by immunohistochemistry that IDOi treatment decreasesβ-catenin levels, increases CD8 T-cell infiltration, and decreases IDO1levels in 4T1 tumors.

FIG. 9C shows that two cycles of IDOi treatment inhibits tumor growth ascompared to placebo in 4T1 tumors that were implanted into Balb/C mice.

FIGS. 10A-C show the efficacy of IDOi (epacadostat), an anti-PD-1antibody (PD-1), and EX M3 administered as single agents (FIG. 10A),combinations of two agents (FIG. 10B), or combinations of three agents(FIG. 10C) in Balb/C mice implanted with 4T1 tumors, with thecombination of all three agents showing tumor regression, as describedin Example 7.

FIGS. 11A-B show the mRNA levels of CD8 (FIG. 11A) and Foxp3 (FIG. 11B)in 4T1 tumors treated with IDOi, anti-PD-1 antibody and/or EX M3 anddemonstrates that only the combination of all three agents significantlyincreased CD8 mRNA levels and significantly decreased Foxp3 mRNA levels.

DEFINITIONS

In order for the present disclosure to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms may be set forth through thespecification. If a definition of a term set forth below is inconsistentwith a definition in an application or patent that is incorporated byreference, the definition set forth in this application should be usedto understand the meaning of the term.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, a reference to “a method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Administer As used herein, “administering” a composition to a subjectmeans to give, apply or bring the composition into contact with thesubject. Administration can be accomplished by any of a number ofroutes, including, for example, topical, oral, subcutaneous,intramuscular, intraperitoneal, intravenous, intrathecal andintradermal.

Antibody: As used herein, the term “antibody” refers to animmunoglobulin or an antigen-binding domain thereof. The term includesbut is not limited to polyclonal, monoclonal, monospecific,polyspecific, non-specific, humanized, human, single-chain, chimeric,synthetic, recombinant, hybrid, mutated, grafted, and in vitro generatedantibodies. The antibody can include a constant region, or a portionthereof, such as the kappa, lambda, alpha, gamma, delta, epsilon and muconstant region genes. For example, heavy chain constant regions of thevarious isotypes can be used, including: IgG₁, IgG₂, IgG₃, IgG₄, IgM,IgA₁, IgA₂, IgD, and IgE. By way of example, the light chain constantregion can be kappa or lambda.

Antigen-Binding Domain: As used herein, the term “antigen-bindingdomain” refers to a part of an antibody molecule that comprises aminoacids responsible for the specific binding between antibody and antigen.For certain antigens, the antigen-binding domain may only bind to a partof the antigen. The part of the antigen that is specifically recognizedand bound by the antibody is referred to as the “epitope” or “antigenicdeterminant.” Antigen-binding domains include Fab (Fragmentantigen-binding); a F(ab′)₂ fragment, a bivalent fragment having two Fabfragments linked by a disulfide bridge at the hinge region; Fv fragment;a single chain Fv fragment (scFv) see e.g., Bird et al. (1988) Science242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883); a Fd fragment having the two V_(H) and C_(H)1 domains;dAb (Ward et al., (1989) Nature 341:544-546), and other antibodyfragments that retain antigen-binding function. The Fab fragment hasV_(H)-C_(H)1 and V_(L)-C_(L) domains covalently linked by a disulfidebond between the constant regions. The F_(v) fragment is smaller and hasV_(H) and V_(L) domains non-covalently linked. To overcome the tendencyof non-covalently linked domains to dissociate, a scF_(v) can beconstructed. The scF_(v) contains a flexible polypeptide that links (1)the C-terminus of V_(H) to the N-terminus of V_(L), or (2) theC-terminus of V_(L) to the N-terminus of V_(H). A 15-mer (Gly₄Ser)₃peptide may be used as a linker, but other linkers are known in the art.These antibody fragments are obtained using conventional techniquesknown to those with skill in the art, and the fragments are evaluatedfor function in the same manner as are intact antibodies.

Antisense strand: A double-stranded nucleic acid inhibitor moleculecomprises two oligonucleotide strands: an antisense strand and a sensestrand. The antisense strand or a region thereof is partially,substantially or fully complementary to a corresponding region of atarget nucleic acid. In addition, the antisense strand of thedouble-stranded nucleic acid inhibitor molecule or a region thereof ispartially, substantially or fully complementary to the sense strand ofthe double-stranded nucleic acid inhibitor molecule or a region thereof.In certain embodiments, the antisense strand may also containnucleotides that are non-complementary to the target nucleic acidsequence. The non-complementary nucleotides may be on either side of thecomplementary sequence or may be on both sides of the complementarysequence. In certain embodiments, where the antisense strand or a regionthereof is partially or substantially complementary to the sense strandor a region thereof, the non-complementary nucleotides may be locatedbetween one or more regions of complementarity (e.g., one or moremismatches). The antisense strand of a double-stranded nucleic acidinhibitor molecule is also referred to as the guide strand.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

β-catenin: As used herein, “β-catenin” refers either to a polypeptide ora nucleic acid sequence encoding such a β-catenin protein. Whenreferring to a polypeptide, “B-catenin” refers to the polypeptide geneproduct of a β-catenin gene/transcript (CTNNB1) (Genbank Accession Nos.NM_001904.3 (human β-catenin transcript variant 1), NM_001098209.1(human β-catenin transcript variant 2), NM_001098210.1 (human β-catenintranscript variant 3), and NM_007614.2 & NM_007614.3 (mouse β-catenin).

Complementary: As used herein, the term “complementary” refers to astructural relationship between two nucleotides (e.g., on two opposingnucleic acids or on opposing regions of a single nucleic acid strand)that permits the two nucleotides to form base pairs with one another.For example, a purine nucleotide of one nucleic acid that iscomplementary to a pyrimidine nucleotide of an opposing nucleic acid maybase pair together by forming hydrogen bonds with one another. In someembodiments, complementary nucleotides can base pair in the Watson-Crickmanner or in any other manner that allows for the formation of stableduplexes. “Fully complementary” or 100% complementarity refers to thesituation in which each nucleotide monomer of a first oligonucleotidestrand or of a segment of a first oligonucleotide strand can form a basepair with each nucleotide monomer of a second oligonucleotide strand orof a segment of a second oligonucleotide strand. Less than 100%complementarity refers to the situation in which some, but not all,nucleotide monomers of two oligonucleotide strands (or two segments oftwo oligonucleotide strands) can form base pairs with each other.“Substantial complementarity” refers to two oligonucleotide strands (orsegments of two oligonucleotide strands) exhibiting 90% or greatercomplementarity to each other. “Sufficiently complementary” refers tocomplementarity between a target mRNA and a nucleic acid inhibitormolecule, such that there is a reduction in the amount of proteinencoded by a target mRNA.

Complementary strand: As used herein, the term “complementary strand”refers to a strand of a double-stranded nucleic acid inhibitor moleculethat is partially, substantially or fully complementary to the otherstrand.

Deoxyribofuranosyl: As used herein, the term “deoxyribofuranosyl” refersto a furanosyl that is found in naturally occurring DNA and has ahydrogen group at the 2′-carbon, as illustrated below:

Deoxyribonucleotide: As used herein, the term “deoxyribonucleotide”refers to a natural nucleotide (as defined herein) or a modifiednucleotide (as defined herein) which has a hydrogen group at the2′-position of the sugar moiety.

dsRNAi inhibitor molecule: As used herein, the term “dsRNAi inhibitormolecule” refers to a double-stranded nucleic acid inhibitor moleculehaving a sense strand (passenger) and antisense strand (guide), wherethe antisense strand or part of the antisense strand is used by theArgonaute 2 (Ago2) endonuclease in the cleavage of a target m RNA.

Duplex: As used herein, the term “duplex,” in reference to nucleic acids(e.g., oligonucleotides), refers to a structure formed throughcomplementary base pairing of two antiparallel sequences of nucleotides.

Excipient: As used herein, the term “excipient” refers to anon-therapeutic agent that may be included in a composition, for exampleto provide or contribute to a desired consistency or stabilizing effect.

Furanosyl: As used herein, the term “furanosyl” refers to a structurecomprising a 5-membered ring with four carbon atoms and one oxygen atom.

Internucleotide linking group: As used herein, the term “internucleotidelinking group” or “internucleotide linkage” refers to a chemical groupcapable of covalently linking two nucleoside moieties. Typically, thechemical group is a phosphorus-containing linkage group containing aphospho or phosphite group. Phospho linking groups are meant to includea phosphodiester linkage, a phosphorodithioate linkage, aphosphorothioate linkage, a phosphotriester linkage, athionoalkylphosphonate linkage, a thionalkylphosphotriester linkage, aphosphoramidite linkage, a phosphonate linkage and/or a boranophosphatelinkage. Many phosphorus-containing linkages are well known in the art,as disclosed, for example, in U.S. Pat. Nos. 3,687,808; 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306;5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555;5,527,899; 5,721,218; 5,672,697 and 5,625,050. In other embodiments, theoligonucleotide contains one or more internucleotide linking groups thatdo not contain a phosphorous atom, such short chain alkyl or cycloalkylinternucleotide linkages, mixed heteroatom and alkyl or cycloalkylinternucleotide linkages, or one or more short chain heteroatomic orheterocyclic internucleotide linkages, including, but not limited to,those having siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; and amide backbones.Non-phosphorous containing linkages are well known in the art, asdisclosed, for example, in U.S. Pat. Nos. 5,034,506; 5,166,315;5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437;5,792,608; 5,646,269 and 5,677,439.

Immune checkpoint molecules: As used herein, the term “immune checkpointmolecule” refers to molecules on immune cells, such as T cells, that areimportant under normal physiological conditions for the maintenance ofself-tolerance (or the prevention of autoimmunity) and the protection ofhost cells and tissue when the immune system responds to a foreignpathogen. Certain immune checkpoint molecules are co-stimulatorymolecules that amplify a signal involved in the T cell response toantigen while certain immune checkpoint molecules are inhibitorymolecules (e.g., CTLA-4 or PD-1) that reduce a signal involved in the Tcell response to antigen.

Modified nucleobase: As used herein, the term “modified nucleobase”refers to any nucleobase that is not a natural nucleobase or a universalnucleobase. Suitable modified nucleobases include diaminopurine and itsderivatives, alkylated purines or pyrimidines, acylated purines orpyrimidines thiolated purines or pyrimidines, and the like. Othersuitable modified nucleobases include analogs of purines andpyrimidines. Suitable analogs include, but are not limited to,1-methyladenine, 2-methyladenine, N6-methyladenine, N6-isopentyladenine,2-methylthio-N6-isopentyladenine, N,N-dimethyladenine, 8-bromoadenine,2-thiocytosine, 3-methylcytosine, 5-methylcytosine, 5-ethylcytosine,4-acetylcytosine, 1-methylguanine, 2-methylguanine, 7-methylguanine,2,2-dimethylguanine, 8-bromoguanine, 8-chloroguanine, 8-am inoguanine,8-methylguanine, 8-thioguanine, 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, 5-ethyluracil, 5-propyluracil,5-methoxyuracil, 5-hydroxymethyluracil, 5-(carboxyhydroxymethyl)uracil,5-(methylaminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil,2-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil,uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester,pseudouracil, 1-methylpseudouracil, queosine, hypoxanthine, xanthine,2-aminopurine, 6-hydroxyaminopurine, nitropyrrolyl, nitroindolyl anddifluorotolyl, 6-thiopurine and 2,6-diaminopurine nitropyrrolyl,nitroindolyl and difluorotolyl. Typically a nucleobase contains anitrogenous base. In certain embodiments, the nucleobase does notcontain a nitrogen atom. See e.g., U.S. Published Patent Application No.20080274462.

Modified nucleoside: As used herein, the term “modified nucleoside”refers to a heterocyclic nitrogenous base in N-glycosidic linkage with asugar (e.g., deoxyribose or ribose or analog thereof) that is not linkedto a phosphate group or a modified phosphate group (as defined herein)and that contains one or more of a modified nucleobase (as definedherein), a universal nucleobase (as defined herein) or a modified sugarmoiety (as defined herein). The modified or universal nucleobases (alsoreferred to herein as base analogs) are generally located at the1′-position of a nucleoside sugar moiety and refer to nucleobases otherthan adenine, guanine, cytosine, thymine and uracil at the 1′-position.In certain embodiments, the modified or universal nucleobase is anitrogenous base. In certain embodiments, the modified nucleobase doesnot contain nitrogen atom. See e.g., U.S. Published Patent ApplicationNo. 20080274462. In certain embodiments, the modified nucleotide doesnot contain a nucleobase (abasic). Suitable modified or universalnucleobases or modified sugars in the context of the present disclosureare described herein.

Modified nucleotide: As used herein, the term “modified nucleotide”refers to a heterocyclic nitrogenous base in N-glycosidic linkage with asugar (e.g., ribose or deoxyribose or analog thereof) that is linked toa phosphate group or a modified phosphate group (as defined herein) andcontains one or more of a modified nucleobase (as defined herein), auniversal nucleobase (as defined herein), or a modified sugar moiety (asdefined herein). The modified or universal nucleobases (also referred toherein as base analogs) are generally located at the 1′-position of anucleoside sugar moiety and refer to nucleobases other than adenine,guanine, cytosine, thymine and uracil at the 1′-position. In certainembodiments, the modified or universal nucleobase is a nitrogenous base.In certain embodiments, the modified nucleobase does not containnitrogen atom. See e.g., U.S. Published Patent Application No.20080274462. In certain embodiments, the modified nucleotide does notcontain a nucleobase (abasic). Suitable modified or universalnucleobases, modified sugar moieties, or modified phosphate groups inthe context of the present disclosure are described herein.

Modified phosphate group: As used herein, the term “modified phosphategroup” refers to a modification of the phosphate group that does notoccur in natural nucleotides and includes non-naturally occurringphosphate mimics as described herein, including phosphate mimics thatinclude a phosphorous atom and anionic phosphate mimics that do notinclude phosphate (e.g. acetate). Modified phosphate groups also includenon-naturally occurring internucleotide linking groups, including bothphosphorous-containing internucleotide linking groups, including, forexample, phosphorothioate, and non-phosphorous containing linkinggroups, as described herein.

Modified sugar moiety: As used herein, a “modified sugar moiety” refersto a substituted sugar moiety (as defined herein) or a sugar analog (asdefined herein).

Natural nucleoside: As used herein, the term “natural nucleoside” refersto a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar(e.g., deoxyribose or ribose or analog thereof). The naturalheterocyclic nitrogenous bases include adenine, guanine, cytosine,uracil and thymine.

Natural nucleotide: As used herein, the term “natural nucleotide” refersto a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar(e.g., ribose or deoxyribose or analog thereof) that is linked to aphosphate group. The natural heterocyclic nitrogenous bases includeadenine, guanine, cytosine, uracil and thymine.

Natural nucleobase: As used herein, the term “natural nucleobase” refersto the five primary, naturally occurring heterocyclic nucleobases of RNAand DNA, i.e., the purine bases: adenine (A) and guanine (G), and thepyrimidine bases: thymine (T), cytosine (C), and uracil (U).

Natural nucleoside: As used herein, the term “natural nucleoside” refersto a natural nucleobase (as defined herein) in N-glycosidic linkage witha natural sugar moiety (as defined herein) that is not linked to aphosphate group.

Natural nucleotide: As used herein, the term “natural nucleotide” refersto a natural nucleobase (as defined herein) in N-glycosidic linkage witha natural sugar moiety (as defined herein) that is linked to a phosphategroup.

Natural sugar moiety: As used herein, the term “natural sugar moiety”refers to a ribofuranosyl (as defined herein) or a deoxyribofuranosyl(as defined herein).

non-T cell inflamed phenotype: As used herein, “non-T cell inflamedphenotype” refers to a tumor microenvironment without a pre-existing Tcell response against the tumor, as evidenced by little to noaccumulation of infiltrating CD8+ T cells in the tumor microenvironment.Typically, the non-T cell inflamed phenotype is also characterized by alimited chemokine profile that does not promote the recruitment andaccumulation of CD8+ T cells in the tumor microenvironment and/or aminimal or absent type I IFN gene signature.

non-Wnt activated disease or disorder. As used herein, a “non-Wntactivated” disease or disorder refers to a disease or disorder that isnot associated with activation of the Wnt/β-catenin pathway. A “non-Wntactivated” disease or disorder includes certain cancer and/orproliferative diseases, conditions, or disorders, including certaincolorectal, desmoid, endometrial, gastric, hepatocellular,hepatoblastoma, kidney (Wilms' tumor), medulloblastoma, melanoma,neuroblastoma, ovarian (endometrioid), pancreatic, pilomatricoma,prostate, renal, thyroid (anaplastic) and uterine (endometrium) cancers.In one embodiment, the “non-Wnt activated” disease or disorder iscolorectal cancer, hepatocellular carcinoma, or melanoma. In oneembodiment, the “non-Wnt activated” disease or disorder isneuroblastoma, renal cancer, or melanoma. It is understood that adisease or disorder, including the cancer and/or proliferative diseaseslisted above, may include both a non-Wnt activated sub-type of thedisease or disorder and a Wnt activated sub-type of the disease ordisorder, consistent with the definition of Wnt activated disease ordisorder provided below.

Nucleic acid inhibitor molecule: As used herein, the term “nucleic acidinhibitor molecule” refers to an oligonucleotide molecule that reducesor eliminates the expression of a target gene wherein theoligonucleotide molecule contains a region that specifically targets asequence in the target gene mRNA. Typically, the targeting region of thenucleic acid inhibitor molecule comprises a sequence that issufficiently complementary to a sequence on the target gene mRNA todirect the effect of the nucleic acid inhibitor molecule to thespecified target gene. The nucleic acid inhibitor molecule may includeribonucleotides, deoxyribonucleotides, and/or modified nucleotides.

Nucleobase: As used herein, the term “nucleobase” refers to a naturalnucleobase (as defined herein), a modified nucleobase (as definedherein), or a universal nucleobase (as defined herein).

Nucleoside: As used herein, the term “nucleoside” refers to a naturalnucleoside (as defined herein) or a modified nucleoside (as definedherein).

Nucleotide: As used herein, the term “nucleotide” refers to a naturalnucleotide (as defined herein) or a modified nucleotide (as definedherein).

Overhang: As used herein, the term “overhang” refers to terminalnon-base pairing nucleotide(s) at either end of either strand of adouble-stranded nucleic acid inhibitor molecule. In certain embodiments,the overhang results from one strand or region extending beyond theterminus of the complementary strand to which the first strand or regionforms a duplex. One or both of two oligonucleotide regions that arecapable of forming a duplex through hydrogen bonding of base pairs mayhave a 5′- and/or 3′-end that extends beyond the 3′- and/or 5′-end ofcomplementarity shared by the two polynucleotides or regions. Thesingle-stranded region extending beyond the 3′- and/or 5′-end of theduplex is referred to as an overhang.

Pharmaceutical composition: As used herein, the term “pharmaceuticalcomposition” comprises a pharmacologically effective amount of aβ-catenin nucleic acid inhibitor molecule or an immunotherapeutic agent,such as an antibody (including, for example, one or more of ananti-CTLA-4, anti-PD-1, or anti-PD-L1 antibody) and a pharmaceuticallyacceptable excipient.

Pharmaceutically acceptable excipient: As used herein, the term“pharmaceutically acceptable excipient” means that the excipient is onethat is suitable for use with humans and/or animals without undueadverse side effects (such as toxicity, irritation, and allergicresponse) commensurate with a reasonable benefit/risk ratio.

Phosphate mimic: As used herein, the term “phosphate mimic” refers to achemical moiety at the 5′-terminal end of an oligonucleotide that mimicsthe electrostatic and steric properties of a phosphate group. Manyphosphate mimics have been developed that can be attached to the 5′-endof an oligonucleotide (see, e.g., U.S. Pat. No. 8,927,513; Prakash etal. Nucleic Acids Res., 2015,43(6):2993-3011). Typically, these5′-phosphate mimics contain phosphatase-resistant linkages. Suitablephosphate mimics include 5′-phosphonates, such as5′-methylenephosphonate (5′-MP) and 5′-(E)-vinylphosphonate (5′-VP) and4′-phosphate analogs that are bound to the 4′-carbon of the sugar moiety(e.g., a ribose or deoxyribose or analog thereof) of the 5′-terminalnucleotide of an oligonucleotide, such as 4′-oxymethylphosphonate,4′-thiomethylphosphonate, or 4′-aminomethylphosphonate, as described inInternational Publication No. WO 2018/045317, which is herebyincorporated by reference in its entirety. In certain embodiments, the4′-oxymethylphosphonate is represented by the formula —O—CH₂—PO(OH)₂ or—O—CH₂—PO(OR)₂, where R is independently selected from H, CH₃, an alkylgroup, or a protecting group. In certain embodiments, the alkyl group isCH₂CH₃. More typically, R is independently selected from H, CH₃, orCH₂CH₃. Other modifications have been developed for the 5′-end ofoligonucleotides (see, e.g., WO 2011/133871).

Potentiate: The term “potentiate” or “potentiating” as used hereinrefers to the ability of one therapeutic agent (e.g., a β-cateninnucleic acid inhibitor molecule) to increase or enhance the therapeuticeffect of another therapeutic agent (e.g., an antagonist of aninhibitory immune checkpoint molecule, such as CTLA-4 or PD-1, or anagonist of a co-stimulatory checkpoint molecule).

Protecting group: As used herein, the term “protecting group” is used inthe conventional chemical sense as a group which reversibly rendersunreactive a functional group under certain conditions of a desiredreaction. After the desired reaction, protecting groups may be removedto deprotect the protected functional group. All protecting groupsshould be removable under conditions which do not degrade a substantialproportion of the molecules being synthesized.

Reduce(s): The term “reduce” or “reduces” as used herein refers to itsmeaning as is generally accepted in the art. With reference to nucleicacid inhibitor molecules, the term generally refers to the reduction inthe expression of a gene, or level of RNA molecules or equivalent RNAmolecules encoding one or more proteins or protein subunits, or activityof one or more proteins or protein subunits, below that observed in theabsence of the nucleic acid inhibitor molecules.

Resistance: The term “resistance” or “resistant” as used in relation toimmunotherapy refers to a cancer and/or proliferative disease, conditionor disorder that does not show a medically significant response toimmunotherapy. As disclosed herein, resistance to immunotherapy can bereversed by reducing β-catenin expression.

Ribofuranosyl: As used herein, the term “ribofuranosyl” refers to afuranosyl that is found in naturally occurring RNA and has a hydroxylgroup at the 2′-carbon, as illustrated below:

Ribonucleotide: As used herein, the term “ribonucleotide” refers to anatural nucleotide (as defined herein) or a modified nucleotide (asdefined herein) which has a hydroxyl group at the 2′-position of thesugar moiety.

Sense strand: A double-stranded nucleic acid inhibitor moleculecomprises two oligonucleotide strands: an antisense strand and a sensestrand. The sense strand or a region thereof is partially, substantiallyor fully complementary to the antisense strand of the double-strandednucleic acid inhibitor molecule or a region thereof. In certainembodiments, the sense strand may also contain nucleotides that arenon-complementary to the antisense strand. The non-complementarynucleotides may be on either side of the complementary sequence or maybe on both sides of the complementary sequence. In certain embodiments,where the sense strand or a region thereof is partially or substantiallycomplementary to the antisense strand or a region thereof, thenon-complementary nucleotides may be located between one or more regionsof complementarity (e.g., one or more mismatches). The sense strand isalso called the passenger strand.

Subject: As used herein, the term “subject” means any mammal, includingmice, rabbits, and humans. In one embodiment, the subject is a human.The terms “individual” or “patient” are intended to be interchangeablewith “subject.”

Substituent or substituted: The terms “substituent” or “substituted” asused herein refer to the replacement of hydrogen radicals in a givenstructure with the radical of a substituent. When more than one positionin any given structure may be substituted with more than onesubstituent, the substituent may be either the same or different atevery position unless otherwise indicated. As used herein, the term“substituted” is contemplated to include all permissible substituentsthat are compatible with organic compounds. The permissible substituentsinclude acyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic substituents of organiccompounds. This disclosure is not intended to be limited in any mannerby the permissible substituents of organic compounds.

Substituted sugar moiety. As used herein, a “substituted sugar moiety”includes furanosyls comprising one or more modifications. Typically, themodifications occur at the 2′-, 3′- , 4′-, or 5′-carbon position of thesugar.

Sugar analog: As used herein, the term “sugar analog” refers to astructure that does not comprise a furanosyl and that is capable ofreplacing the naturally occurring sugar moiety of a nucleotide, suchthat the resulting nucleotide is capable of (1) incorporation into anoligonucleotide and (2) hybridization to a complementary nucleotide.Such structures typically include relatively simple changes to thefuranosyl, such as rings comprising a different number of atoms (e.g.,4, 6, or 7-membered rings); replacement of the oxygen of the furanosylwith a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both achange in the number of atoms and a replacement of the oxygen. Suchstructures may also comprise substitutions corresponding with thosedescribed for substituted sugar moieties. Sugar analogs also includemore complex sugar replacements (e.g., the non-ring systems of peptidenucleic acid). Sugar analogs include without limitation morpholinos,cyclohexenyls and cyclohexitols.

Sugar moiety: As used herein, the term “sugar moiety” refers to anatural sugar moiety or a modified sugar moiety of a nucleotide ornucleoside.

Target site: As used herein, the term “target site” “target sequence,”“target nucleic acid”, “target region,” “target gene” are usedinterchangeably and refer to a RNA or DNA sequence that is “targeted,”e.g., for cleavage mediated by a dsRNAi inhibitor molecule that containsa sequence within its guide/antisense region that is partially,substantially, or perfectly or sufficiently complementary to that targetsequence.

T cell-inflamed tumor phenotype: As used herein, “T cell-inflamedphenotype” refers to a tumor microenvironment with a pre-existing T cellresponse against the tumor, as evidenced by an accumulation ofinfiltrating CD8+ T cells in the tumor microenvironment. Typically, theT cell-inflamed phenotype is also characterized by a broad chemokineprofile capable of recruiting CD8+ T cells to the tumor microenvironment(including CXCL9 and/or CXCL10) and/or a type I IFN gene signature.

Therapeutically effective amount: As used herein, a “therapeuticallyeffective amount” or “pharmacologically effective amount” refers to thatamount of a β-catenin nucleic acid inhibitor molecule or animmunotherapeutic agent, such as an antibody (including, for example,one or more of an anti-CTLA-4, anti-PD-1, or anti-PD-L1 antibody)effective to produce the intended pharmacological, therapeutic orpreventive result.

Universal nucleobase: As used herein, a “universal nucleobase” refers toa base that can pair with more than one of the bases typically found innaturally occurring nucleic acids and can thus substitute for suchnaturally occurring bases in a duplex. The base need not be capable ofpairing with each of the naturally occurring bases. For example, certainbases pair only or selectively with purines, or only or selectively withpyrimidines. The universal nucleobase may base pair by forming hydrogenbonds via Watson-Crick or non-Watson-Crick interactions (e.g., Hoogsteeninteractions). Representative universal nucleobases include inosine andits derivatives.

Wnt activated disease or disorder. As used herein, a “Wnt activated”disease or disorder refers to a disease or disorder that is associatedwith an activated Wnt/β-catenin pathway. A “Wnt-associated” disease ordisorder includes cancer and/or proliferative diseases, conditions, ordisorders, including colorectal, desmoid, endometrial, gastric,hepatocellular, hepatoblastoma, kidney (Wilms' tumor), medulloblastoma,melanoma, ovarian (endometrioid), pancreatic, pilomatricoma, prostate,thyroid (anaplastic) and uterine (endometrium) cancers. In oneembodiment, the “Wnt activated” disease or disorder is colorectalcancer, hepatocellular carcinoma, or melanoma. It is understood that adisease or disorder, including the cancer and/or proliferative diseaseslisted above, may include both a Wnt activated version of the disease ordisorder and a non-Wnt activated version of the disease or disorder,consistent with the definition of non-Wnt activated disease or disorderprovided above.

Wnt/β-catenin pathway: As used herein the “Wnt/β-catenin pathway” refersto a molecular signaling pathway in cells that is mediated through acombination of Wnt ligands, receptors, and co-receptors, which initiatea downstream signaling pathway that involves β-catenin (see e.g., FIG.6). In the absence of Wnt signaling, β-catenin is targeted fordegradation via ubiquitination in the cellular cytoplasm. In thepresence of Wnt ligand and Wnt signaling, β-catenin is stabilized andtravels to the cell nucleus where it can interact with transcriptionfactors, such as T cell transcription factor (TCF) and lymphoid enhancedtranscription factor (LEF), and activate gene transcription.Deregulation and activation of the Wnt/β-catenin pathway is most oftencaused by mutations in the β-catenin gene or the gene encodingadenomatous polyposis coli (APC), which negatively regulates β-cateninfunction, but can also be caused by a mutation in a gene encoding othercomponents of the Wnt/β-catenin pathway, such as Axin, LEF, and ICAT.

DETAILED DESCRIPTION

This application provides a potent, optimized double-stranded β-cateninnucleic acid inhibitor molecule having a unique pattern of modifiednucleotides. As shown in the examples, changing the modificationpatterns of the nucleotides in the sense and antisense strands of adouble-stranded nucleic acid molecule can result in improved properties,including reduced immunogenicity and improved reduction of β-cateninmRNA expression in tumor cells, even when the nucleotide sequences ofthe sense and antisense strands are identical. Also provided are methodsof using the optimized, double-stranded β-catenin nucleic acid inhibitormolecule and compositions comprising the same to reduce the level orexpression of the β-catenin gene in vitro or in vivo, including methodsand compositions for treating cancer, including cancer that is notresponsive to immunotherapy (e.g., blockade of immune checkpointmolecules).

Optimized β-Catenin Nucleic Acid Inhibitor Molecule

The double-stranded β-catenin nucleic acid inhibitor molecule describedherein comprises a unique pattern of nucleotide modifications andpossesses improved properties relative to β-catenin nucleic acidinhibitor molecules that do not share the same pattern of nucleotidemodifications. In certain embodiments, the sense strand of the optimizedβ-catenin nucleic acid inhibitor molecule comprises the nucleic acid ofSEQ ID NO: 11. In certain embodiments, the sense strand of the optimizedβ-catenin nucleic acid inhibitor molecule consists of the nucleic acidof SEQ ID NO: 11. In certain embodiments, the antisense strand of theoptimized β-catenin nucleic acid inhibitor molecule comprises thenucleic acid of SEQ ID NO: 12. In certain embodiments, the antisensestrand of the optimized β-catenin nucleic acid inhibitor moleculeconsists of the nucleic acid of SEQ ID NO: 12. In one embodiment, thesense strand consists of the nucleic acid of SEQ ID NO: 11 and theantisense strand consists of the nucleic acid of SEQ ID NO: 12. In oneembodiment, the sense strand comprises the nucleic acid of SEQ ID NO: 11and the antisense strand comprises the nucleic acid of SEQ ID NO: 12.

In certain embodiments, the optimized β-catenin nucleic acid inhibitormolecule is a double-stranded nucleic acid inhibitor molecule comprisinga sense strand and an antisense strand, wherein the sense strandcomprises or consists of the nucleic acid of SEQ ID NO: 11 and theantisense strand comprises or consists of the nucleic acid of SEQ ID NO:12 and the sense strand and antisense strand together form a duplexregion of 26 base pairs, and wherein the antisense strand includes 2single-stranded nucleotides at its 3′ terminus and 10 single-strandednucleotides at its 5′ terminus.

A schematic of this optimized β-catenin nucleic acid inhibitor moleculeis set forth in FIG. 2A (“EX M3”), showing the sequences of the senseand antisense strands, the region of complementarity between the senseand antisense strands, and the unique pattern of modified nucleotides.

The unique pattern of modified nucleotides is also set forth in SEQ IDNO: 11 and SEQ ID NO: 12. As set forth in SEQ ID NO: 11 and SEQ ID NO:12, the sugar moiety of most of the nucleotides in the modifiedβ-catenin nucleic acid inhibitor molecule is modified with either a 2′-For a 2′-OCH₃. Specifically, for the sense strand (SEQ ID NO: 11), thesugar moiety of each of nucleotides 1, 3, 7, 9-11, 13, 15, 19, and 20 ismodified with a 2′-F, and the sugar moiety of each of nucleotides 2,4-6, 8, 12, 14, 16-18, and 24 of is modified with a 2′-OCH₃, as depictedin FIG. 2A. For the antisense strand (SEQ ID NO: 12), the sugar moietyof each of nucleotides 2, 6, 19, 21, 23, 25, 30, 31, 33, and 35 ismodified with a 2′-F, and the sugar moiety of each of nucleotides 1,3-5, 7-9, 11-15, 20, 22, 24, 26, 32, 34, and 36-38 is modified with a2′-OCH₃, as depicted in FIG. 2. The remaining nucleotides in theoptimized β-catenin nucleic acid inhibitor molecule are not modifiedwith either a 2′-For a 2′-OCH₃. More specifically, for the sense strand(SEQ ID NO: 11), nucleotides 25 and 26 are deoxyribonucleotides andnucleotides 21-23 are ribonucleotides. Typically, nucleotides 25 and 26are natural deoxyribonucleotides and nucleotides 21-23 are naturalribonucleotides. For the antisense strand (SEQ ID NO: 12), nucleotide 10is a deoxyribonucleotide and nucleotides 16-18 and 27-29 areribonucleotides. Typically, nucleotide 10 is a naturaldeoxyribonucleotide and nucleotides 16-18 and 27-29 are naturalribonucleotides.

In one embodiment, the optimized β-catenin nucleic acid inhibitormolecule is a double-stranded nucleic acid inhibitor molecule comprisinga sense strand and an antisense strand;

wherein the sense strand comprises or consists of the nucleic acidsequence of SEQ ID NO: 13 (agaauacaaaugauguagaaacagcc) and the antisensestrand comprises or consists of the nucleic acid sequence of SEQ ID NO:14 (uagcuaucgtggcuguuucuacaucauuuguauucugc) and the sense strand andantisense strand together form a duplex region of 26 base pairs and theantisense strand includes 2 single-stranded nucleotides at its 3′terminus and 10 single-stranded nucleotides at its 5′ terminus;

wherein the sugar moiety of each of nucleotides 1, 3, 7, 9-11, 13, 15,19, and 20 of SEQ ID NO: 13 is modified with a 2′-F, the sugar moiety ofeach of nucleotides 2, 4-6, 8, 12, 14, 16-18, and 24 of SEQ ID NO: 13 ismodified with a 2′-OCH₃, nucleotides 25 and 26 of SEQ ID NO: 13 arenatural deoxyribonucleotides, and nucleotides 21-23 of SEQ ID NO: 13 arenatural ribonucleotides;

wherein the sugar moiety of each of nucleotides 2, 6, 19, 21, 23, 25,30, 31, 33, and 35 of SEQ ID NO: 14 is modified with a 2′-F, the sugarmoiety of each of nucleotides 1, 3-5, 7-9, 11-15, 20, 22, 24, 26, 32,34, and 36-38 of SEQ ID NO: 14 is modified with a 2′-OCH₃, nucleotide 10of SEQ ID NO: 14 is a natural deoxyribonucleotide, and nucleotides 16-18and 27-29 of SEQ ID NO: 14 are natural ribonucleotides.

A related aspect is directed to an oligonucleotide comprising orconsisting of the nucleic acid of SEQ ID NO: 11. Another aspect isdirected to an oligonucleotide comprising or consisting of the nucleicacid of SEQ ID NO: 12.

Also provided is an oligonucleotide comprising or consisting of thenucleic acid of SEQ ID NO: 13, wherein the sugar moiety of each ofnucleotides 1, 3, 7, 9-11, 13, 15, 19, and 20 is modified with a 2′-F;the sugar moiety of each of nucleotides 2, 4-6, 8, 12, 14, 16-18, and 24is modified with a 2′-OCH₃; nucleotides 25 and 26 aredeoxyribonucleotides; and nucleotides 21-23 are ribonucleotides.Typically, nucleotides 25 and 26 are natural deoxyribonucleotides andnucleotides 21-23 are natural ribonucleotides.

Also provided is an oligonucleotide comprising or consisting of thenucleic acid of SEQ ID NO: 14, wherein the sugar moiety of each ofnucleotides 2, 6, 19, 21, 23, 25, 30, 31, 33, and 35 is modified with a2′-F; the sugar moiety of each of nucleotides 1, 3-5, 7-9, 11-15, 20,22, 24, 26, 32, 34, and 36-38 is modified with a 2′-OCH₃; nucleotide 10is a deoxyribonucleotide; and nucleotides 16-18 and 27-29 areribonucleotides. Typically, nucleotide 10 is a naturaldeoxyribonucleotide and nucleotides 16-18 and 27-29 are naturalribonucleotides.

Other Modifications

As described herein, the optimized β-catenin nucleic acid inhibitormolecule contains a unique pattern of nucleotide modifications,including a unique pattern of 2′-F and 2′-OMe modifications at the2′-carbon of the sugar moiety of certain nucleotides, as set forth inSEQ ID NO: 11 and SEQ ID NO: 12.

In certain embodiments, the nucleotides of the optimized β-cateninnucleic acid inhibitor molecule contain one or more additionalmodifications that occur at other parts of the nucleotide, including thenucleobase, the phosphate group, or other parts of the sugar moiety.

For example, in certain embodiments, the ring structure of the sugarmoiety can be modified, including, but not limited to, the modified ringstructure present in Locked Nucleic Acids (“LNA”) (see, e.g., Koshkin etal. (1998), Tetrahedron, 54,3607-3630); Bridged Nucleic acids (“BNA”)(see, e.g., U.S. Pat. No. 7,427,672 and Mitsuoka et al. (2009), NucleicAcids Res., 37(4):1225-38); and Unlocked Nucleic Acids (“UNA”) (see,e.g., Snead et al. (2013), Molecular Therapy—Nucleic Acids, 2,e103(doi:10.1038/mtna.2013.36)). Additional modifications can also occur at otherparts of the sugar moiety of the nucleotide, such as the 5′-carbon, asdescribed herein.

In certain embodiments, the optimized β-catenin nucleic acid inhibitormolecule can also include one or more modified nucleobases other thanadenine, guanine, cytosine, thymine and uracil at the 1′-position, asknown in the art and as described herein. In certain embodiments, themodified or universal nucleobase is a nitrogenous base. In certainembodiments, the modified nucleobase does not contain nitrogen atom. Seee.g., U.S. Published Patent Application No. 20080274462. In certainembodiments, the modified nucleotide does not contain a nucleobase(abasic). A typical example of a modified nucleobase is5′-methylcytosine.

In certain embodiments, the optimized β-catenin nucleic acid inhibitormolecule can also include one or more modified phosphate groups. Amodified phosphate group refers to a modification of the phosphate groupthat does not occur in natural nucleotides and includes non-naturallyoccurring phosphate mimics as described herein, including phosphatemimics that include a phosphorous atom and anionic phosphate mimics thatdo not include phosphate (e.g. acetate). Modified phosphate groups alsoinclude non-naturally occurring internucleotide linking groups,including both phosphorous-containing internucleotide linking groups andnon-phosphorous containing linking groups, as described herein.Typically, the optimized β-catenin nucleic acid inhibitor moleculecontains one or more phosphorous-containing internucleotide linkinggroups, as described herein. In other embodiments, one or more of theinternucleotide linking groups of the nucleic acid inhibitor molecule isa non-phosphorus containing linkage. In certain embodiments, theoptimized β-catenin nucleic acid inhibitor molecule contains one or morephosphorous-containing internucleotide linking groups and one or morenon-phosphorous containing internucleotide linking groups, as describedherein.

The 5′-end of the nucleic acid inhibitor molecule can include a naturalsubstituent, such as a hydroxyl or a phosphate group. In certainembodiments, a hydroxyl group is attached to the 5′-terminal end of thesense or antisense strand of the nucleic acid inhibitor molecule. Incertain embodiments, a phosphate group is attached to the 5′-terminalend of the nucleic acid inhibitor molecule. Typically, the phosphate isadded to a monomer prior to oligonucleotide synthesis. In otherembodiments, 5′-phosphorylation is accomplished naturally after anucleic acid inhibitor molecule is introduced into the cytosol, forexample, by a cytosolic Clp1 kinase. In some embodiments, the5′-terminal phosphate is a phosphate group, such as 5′-monophosphate[(HO)₂(O)P—O-5′], 5′-diphosphate [(HO)₂(O)P—O—P(HO)(O)—O-5′] or a5′-triphosphate[(HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)-0-5′].

In certain embodiments one or two nucleotides of the optimized β-cateninnucleic acid inhibitor molecule are reversibly modified with aglutathione-sensitive moiety. Typically, the glutathione-sensitivemoiety is located at the 2′-carbon of the sugar moiety and comprises adisulfide bridge or a sulfonyl group. In certain embodiment, theglutathione-sensitive moiety is compatible with phosphoramiditeoligonucleotide synthesis methods, as described, for example, inInternational Application No. PCT/US2017/048239, which is herebyincorporated by reference in its entirety. In certain embodiments, morethan two nucleotides of the optimized β-catenin nucleic acid inhibitormolecule are reversibly modified with a glutathione-sensitive moiety. Incertain embodiments, most of the nucleotides are reversibly modifiedwith a glutathione-sensitive moiety. In certain embodiments, all orsubstantially all the nucleotides of the optimized β-catenin nucleicacid inhibitor molecule are reversibly modified with aglutathione-sensitive moiety.

The at least one glutathione-sensitive moiety is typically located atthe 5′- or 3′-terminal nucleotide of the passenger strand or the guidestrand of a double-stranded nucleic acid inhibitor molecule. However,the at least one glutathione-sensitive moiety may be located at anynucleotide of interest in the optimized β-catenin nucleic acid inhibitormolecule.

Methods of Reducing β-Catenin Expression

The optimized nucleic acid inhibitor molecule, as described herein, canbe used in methods of reducing β-catenin mRNA expression. Typically, themethod of reducing β-catenin mRNA expression comprises administering theoptimized nucleic acid inhibitor molecule, as described herein, to asample or to a subject in need thereof in an amount sufficient to reduceexpression of the β-catenin gene. The methods may be carried out invitro or in vivo.

The level or activity of a β-catenin RNA can be determined by a suitablemethod now known in the art or that is later developed. It can beappreciated that the method used to measure a target RNA and/or the“expression” of a target gene can depend upon the nature of the targetgene and its encoded RNA. For example, where the target β-catenin RNAsequence encodes a protein, the term “expression” can refer to a proteinor the β-catenin RNA/transcript derived from the β-catenin gene (eithergenomic or of exogenous origin). In such instances the expression of thetarget β-catenin RNA can be determined by measuring the amount ofβ-catenin RNA/transcript directly or by measuring the amount ofβ-catenin protein. Protein can be measured in protein assays such as bystaining or immunoblotting or, if the protein catalyzes a reaction thatcan be measured, by measuring reaction rates. All such methods are knownin the art and can be used. Where target β-catenin RNA levels are to bemeasured, art-recognized methods for detecting RNA levels can be used(e.g., RT-PCR, Northern Blotting, etc.). In targeting β-catenin RNAs,measurement of the efficacy of the nucleic acid inhibitor molecule inreducing levels of β-catenin RNA or protein in a subject, tissue, incells, either in vitro or in vivo, or in cell extracts can also be usedto determine the extent of reduction of β-catenin-associated phenotypes(e.g., disease or disorders, e.g., cancer or tumor formation, growth,metastasis, spread, etc.), as disclosed, for example, in InternationalApplication No. PCT/US2017/022510. The above measurements can be made oncells, cell extracts, tissues, tissue extracts or other suitable sourcematerial.

Pharmaceutical Compositions

The present disclosure provides pharmaceutical compositions comprising atherapeutically effective amount of the optimized β-catenin nucleic acidinhibitor molecule, as described herein, and a pharmaceuticallyacceptable excipient.

These pharmaceutical compositions may be sterilized by conventionalsterilization techniques, or may be sterile filtered. The resultingaqueous solutions may be packaged for use as is, or lyophilized, thelyophilized preparation being combined with a sterile aqueous excipientprior to administration. The pH of the preparations typically will bebetween 3 and 11, more preferably between 5 and 9 or between 6 and 8,and most preferably between 7 and 8, such as 7 to 7.5.

The pharmaceutical compositions of the present disclosure are appliedfor therapeutic use. Thus, one aspect of the disclosure provides apharmaceutical composition, which may be used to treat a subjectincluding, but not limited to, a human suffering from a disease or acondition by administering to said subject a therapeutically effectiveamount of a pharmaceutical composition of the present disclosure.Typically, the disease or condition is cancer, as described herein.

In certain embodiments, the present disclosure features the use of atherapeutically effective amount of a pharmaceutical composition asdescribed herein for the manufacture of a medicament for treatment of asubject in need thereof. Typically, the subject has cancer, as describedherein.

Pharmaceutically-Acceptable Excipients

The pharmaceutically-acceptable excipients useful in this disclosure areconventional. Remington's Pharmaceutical Sciences, by E. W. Martin, MackPublishing Co., Easton, Pa., 15th Edition (1975), describes compositionsand formulations suitable for pharmaceutical delivery of one or moretherapeutic compositions. Some examples of materials which can serve aspharmaceutically-acceptable excipients include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; malt; gelatin; excipients, suchas cocoa butter and suppository waxes; oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; buffering agents, such as magnesium hydroxide and aluminumhydroxide; (isotonic saline; Ringer's solution); ethyl alcohol; pHbuffered solutions; polyols, such as glycerol, propylene glycol,polyethylene glycol, and the like; and other non-toxic compatiblesubstances employed in pharmaceutical formulations.

Dosage Forms

The pharmaceutical compositions may be formulated with conventionalexcipients for any intended route of administration, which may beselected according to ordinary practice.

In one embodiment, the pharmaceutical composition contains the optimizedβ-catenin nucleic acid inhibitor molecule, as described herein, and issuitable for parenteral administration. The pharmaceutical compositionoptionally contains an immunotherapeutic agent, such as an antagonist ofan inhibitory immune checkpoint molecule (e.g., one or more of ananti-CTLA-4, anti-PD-1, or anti-PD-L1 antibody) or an agonist of aco-stimulatory checkpoint molecule. Typically, the pharmaceuticalcompositions of the present disclosure that contain oligonucleotides areformulated in liquid form for parenteral administration, for example, bysubcutaneous, intramuscular, intravenous or epidural injection.

Dosage forms suitable for parenteral administration typically includeone or more suitable vehicles for parenteral administration including,by way of example, sterile aqueous solutions, saline, low molecularweight alcohols such as propylene glycol, polyethylene glycol, vegetableoils, gelatin, fatty acid esters such as ethyl oleate, and the like. Theparenteral formulations may contain sugars, alcohols, antioxidants,buffers, bacteriostats, solutes which render the formulation isotonicwith the blood of the intended recipient or suspending or thickeningagents. Proper fluidity can be maintained, for example, by the use ofsurfactants. Liquid formulations can be lyophilized and stored for lateruse upon reconstitution with a sterile injectable solution.

The pharmaceutical compositions may also be formulated for other routesof administration including topical or transdermal administration,rectal or vaginal administration, ocular administration, nasaladministration, buccal administration, or sublingual administrationusing well known techniques.

Delivery Agents

The optimized β-catenin nucleic acid inhibitor molecule, as describedherein, may be admixed, encapsulated, conjugated or otherwise associatedwith other molecules, molecule structures or mixtures of compounds,including, for example, liposomes and lipids such as those disclosed inU.S. Pat. Nos. 6,815,432, 6,586,410, 6,858,225, 7,811,602, 7,244,448 and8,158,601; polymeric materials such as those disclosed in U.S. Pat. Nos.6,835,393, 7,374,778, 7,737,108, 7,718,193, 8,137,695 and U.S. PublishedPatent Application Nos. 2011/0143434, 2011/0129921, 2011/0123636,2011/0143435, 2011/0142951, 2012/0021514, 2011/0281934, 2011/0286957 and2008/0152661; capsids, capsoids, or receptor targeted molecules forassisting in uptake, distribution or absorption.

In certain embodiments, the optimized β-catenin nucleic acid inhibitormolecule is formulated in a lipid nanoparticle (LNP). Lipid-nucleic acidnanoparticles typically form spontaneously upon mixing lipids withnucleic acid to form a complex. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture can be optionallyextruded through a polycarbonate membrane (e.g., 100 nm cut-off) using,for example, a thermobarrel extruder, such as LIPEX° Extruder (NorthernLipids, Inc). To prepare a lipid nanoparticle for therapeutic use, itmay desirable to remove solvent (e.g., ethanol) used to form thenanoparticle and/or exchange buffer, which can be accomplished by, forexample, dialysis or tangential flow filtration. Methods of making lipidnanoparticles containing nucleic acid interference molecules are knownin the art, as disclosed, for example in U.S. Published PatentApplication Nos. 2015/0374842 and 2014/0107178.

In certain embodiments, the LNP comprises a core lipid componentcomprising a cationic liposome and a pegylated lipid. The LNP canfurther comprise one or more envelope lipids, such as a cationic lipid,a structural or neutral lipid, a sterol, a pegylated lipid, or mixturesthereof.

Cationic lipids for use in LNPs are known in the art, as discussed forexample in U.S. Published Patent Application Nos. 2015/0374842 and2014/0107178. Typically, the cationic lipid is a lipid having a netpositive charge at physiological pH. In certain embodiments, thecationic liposome is DODMA, DOTMA, DL-048, or DL-103. In certainembodiments the structural or neutral lipid is DSPC, DPPC or DOPC. Incertain embodiments, the sterol is cholesterol. In certain embodiments,the pegylated lipid is DMPE-PEG, DSPE-PEG, DSG-PEG, DMPE-PEG2K,DSPE-PEG2K, DSG-PEG2K, or DSG-MPEG. In one embodiment, the cationiclipid is DL-048, the pegylated lipid is DSG-MPEG and the one or moreenvelope lipids are DL-103, DSPC, cholesterol, and DSPE-MPEG. See e.g.,FIG. 5, showing one non-limiting embodiment of a LNP that can used toformulate the optimized β-catenin nucleic acid inhibitor molecule.

In certain embodiments, the optimized β-catenin nucleic acid inhibitormolecule is covalently conjugated to a ligand that directs delivery ofthe oligonucleotide to a tissue of interest. Many such ligands have beenexplored. See, e.g., Winkler, Ther. Deliv. 4(7): 791-809 (2013). Forexample, the optimized β-catenin nucleic acid inhibitor molecule can beconjugated to one or more sugar ligand moieties (e.g.,N-acetylgalactosamine (GaINAc)) to direct uptake of the oligonucleotideinto the liver. See, e.g., U.S. Pat. Nos. 5,994,517; 5,574,142; WO2016/100401. Typically, the optimized β-catenin nucleic acid inhibitormolecule is conjugated to three or four sugar ligand moieties. Otherligands that can be used include, but are not limited to,mannose-6-phosphate, cholesterol, folate, transferrin, and galactose(for other specific exemplary ligands see, e.g., WO 2012/089352).

Methods of Administration/Treatment

One embodiment is directed to a method of treating aβ-catenin-associated disorder, comprising administering to a subject apharmaceutical composition comprising a therapeutically effective amountof the optimized β-catenin nucleic acid inhibitor molecule, as describedherein. In certain embodiments, the β-catenin-associated disorder is acancer.

Non-limiting examples of such cancers include bilary tract cancer,bladder cancer, transitional cell carcinoma, urothelial carcinoma, braincancer, gliomas, astrocytomas, breast carcinoma, metaplastic carcinoma,cervical cancer, cervical squamous cell carcinoma, rectal cancer,colorectal carcinoma, colon cancer, hereditary nonpolyposis colorectalcancer, colorectal adenocarcinomas, gastrointestinal stromal tumors(GISTs), endometrial carcinoma, endometrial stromal sarcomas, esophagealcancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma,ocular melanoma, uveal melanoma, gallbladder carcinomas, gallbladderadenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma,transitional cell carcinoma, urothelial carcinomas, wilms tumor,leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia(AML), chronic lymphocytic (CLL), chronic myeloid (CML), chronicmyelomonocytic (CMML), liver cancer, liver carcinoma, hepatoma,hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, Lungcancer, non-small cell lung cancer (NSCLC), mesothelioma, B-celllymphomas, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, Mantlecell lymphoma, T cell lymphomas, non-Hodgkin lymphoma, precursorT-lymphoblastic lymphoma/leukemia, peripheral T cell lymphomas, multiplemyeloma, nasopharyngeal carcinoma (NPC), neuroblastoma, oropharyngealcancer, oral cavity squamous cell carcinomas, osteosarcoma, ovariancarcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma,pseudopapillary neoplasms, acinar cell carcinomas. Prostate cancer,prostate adenocarcinoma, skin cancer, melanoma, malignant melanoma,cutaneous melanoma, small intestine carcinomas, stomach cancer, gastriccarcinoma, gastrointestinal stromal tumor (GIST), uterine cancer, oruterine sarcoma. In certain embodiments, the present disclosure featuresmethods of treating liver cancer, liver carcinoma, hepatoma,hepatocellular carcinoma, cholangiocarcinoma and hepatoblastoma. Incertain embodiments of the treatment methods, the cancer is colorectalcancer, hepatocellular carcinoma, or melanoma. In certain embodiments ofthe treatment methods, the cancer is a melanoma, a neuroblastoma, or arenal cancer.

In certain embodiments, the pharmaceutical compositions disclosed hereinmay be useful for the treatment or prevention of symptoms related to aWnt activated disease or disorder, such as cancer. In other embodiments,the pharmaceutical compositions disclosed herein may be useful for thetreatment or prevention of symptoms related to a non-Wnt activateddisease or disorder, such as cancer.

In some embodiments, the cancer is associated with an activatedWnt/β-catenin pathway. In other embodiments, the cancer is a non-Wntactivated cancer. In certain embodiments, the subject has beenidentified as having a non-Wnt activated cancer before administering theoptimized β-catenin nucleic acid inhibitor molecule. The subject may beidentified as having a non-Wnt activated cancer using any methodavailable to the skilled artisan. Typically, however, a sample from thesubject is analyzed to determine if the subject has a non-Wnt activatedcancer. In certain embodiments, the sample comprises tissue, cells,blood, or urine. In certain embodiments, the sample is analyzed for oneor more biomarkers associated with an activated Wnt/β-catenin pathway,an inactive Wnt/β-catenin pathway and/or a non-T cell inflamedphenotype. Any appropriate biomarker can be analyzed, including, but notlimited to nucleic acids (e.g., mRNA), proteins, and peptides using anysuitable assay or technique. In certain embodiments, the biomarker is agene mutation that is associated with an activated Wnt/β-cateninpathway, such as a mutation in a gene encoding β-catenin or APC or oneor more other components involved in the Wnt/β-catenin pathway, such as,Axin, LEF, and ICAT.

In certain embodiments, the method of treating cancer further comprisesadministering a therapeutically effective amount of an immunotherapeuticagent. In some embodiments, the immunotherapeutic agent is as anantagonist of an inhibitory immune checkpoint molecule or an agonist ofa co-stimulatory checkpoint molecule. In certain embodiments, theantagonist of an inhibitory immune checkpoint molecule is ananti-CTLA-4, anti-PD-1, anti-PD-L1 antibody, or a combination ofthereof.

In certain embodiments, the cancer is resistant to immunotherapy, butthe resistance to immunotherapy can be reversed by administering theimmunotherapy in combination with a β-catenin nucleic acid inhibitormolecule, such as the optimized β-catenin nucleic acid inhibitormolecule described herein. Typically, cancer that is not responsive toimmunotherapy is characterized by a non-T cell inflamed phenotype (alsoknown as cold or non-inflamed tumors), with little to no infiltratingCD8+ T cells in the tumor microenvironment. Reducing β-cateninexpression can convert a cold or non-inflamed tumor into a hot orinflamed tumor and potentiate the effect of immunotherapy, even intumors that do not have an activated Wnt/β-catenin pathway. In otherwords, by combining a β-catenin inhibitor with immunotherapy, it ispossible to treat cold or non-inflamed tumors that normally do notrespond to immunotherapy. This combination therapy approach has beenshown to potently inhibit tumor growth in vivo across a broad variety ofcancers, including cancers with and without an activated Wnt/β-cateninpathway, as described for example, in U.S. Provisional Application No.62/477,783, which is hereby incorporated by reference in its entirety.In certain embodiments, the cancer is a non-Wnt activated cancer. Incertain embodiments, the cancer is a Wnt activated cancer.

In some embodiments, the present disclosure provides a method ofpotentiating an in vivo immune response against a cancer, comprisingadministering to a subject having cancer the optimized β-catenin nucleicacid inhibitor molecule, as described herein, in an amount sufficient topotentiate the therapeutic effect of immunotherapy against the cancer orotherwise render the cancer susceptible to the immunotherapy. Typically,prior to administering the optimized β-catenin nucleic acid inhibitormolecule, the cancer is associated with a non-T cell inflamed phenotypethat is resistant to immunotherapy and administering the modifiedβ-catenin nucleic acid inhibitor molecule converts the non-T cellinflamed phenotype into a T cell-inflamed phenotype, such that thecancer becomes responsive to immunotherapy. In certain embodiments, thecancer that is resistant to immunotherapy is a Wnt activated cancer. Inother embodiments, the cancer that is resistant to immunotherapy is anon-Wnt activated cancer.

Dosing and Schedule

Typically, the optimized β-catenin nucleic acid inhibitor molecule isadministered parenterally (such as via intravenous, intramuscular, orsubcutaneous administration). In other embodiments, the pharmaceuticalcomposition is delivered via local administration or systemicadministration. However, the pharmaceutical compositions disclosedherein may also be administered by any method known in the art,including, for example, buccal, sublingual, rectal, vaginal,intraurethral, topical, intraocular, intranasal, and/or intraauricular,which administration may include tablets, capsules, granules, aqueoussuspensions, gels, sprays, suppositories, salves, ointments, or thelike.

In certain embodiments, the optimized β-catenin nucleic acid inhibitormolecule is administered at a dosage of 20 micrograms to 10 milligramsper kilogram body weight of the recipient per day, 100 micrograms to 5milligrams per kilogram, 0.25 milligrams to 5.0 milligrams per kilogram,or 0.5 to 3.0 milligrams per kilogram. Typically, the optimizedβ-catenin nucleic acid inhibitor molecule is administered at a dosage ofabout 0.25 to 2.0 milligrams per kilogram body weight of the recipientper day.

A pharmaceutical composition of the instant disclosure may beadministered every day, or intermittently. For example, intermittentadministration of the optimized β-catenin nucleic acid inhibitormolecule may be administration one to six days per week, one to six daysper month, once weekly, once every other week, once monthly, once everyother month, or once or twice per year or divided into multiple yearly,monthly, weekly, or daily doses. Typically, the optimized β-cateninnucleic acid inhibitor molecule is administered every week or every twoweeks. In some embodiments, intermittent dosing may mean administrationin cycles with the initial optimized β-catenin nucleic acid inhibitormolecule or immunotherapeutic agent administration followed by a restperiod with no administration for up to one week, up to one month, up totwo months, up to three months or up to six months or more) or it maymean administration on alternate days, weeks, months or years.

When combined with an immunotherapeutic agent, the β-catenin nucleicacid inhibitor molecule is typically administered separately from, andon a different schedule than, the immunotherapeutic agent.Pharmaceutical compositions containing the immunotherapeutic agent aretypically administered intravenously. For example, when used as a singleagent, ipilimumab (anti-CTLA-4 antibody) is administered intravenouslyover 90 minutes at a recommended dose of 3 mg/kg every 3 weeks for atotal of 4 doses. Similarly, when used as a single agent, nivolumab(anti-PD-1 antibody), is administered intravenously at a recommendeddose of 240 mg (or 3 mg/kg) over 60 minutes every 2 weeks. Whennivolumab is administered in combination with ipilimumab, therecommended dose of nivolumab is 1 mg/kg administered intravenously over60 minutes, followed by ipilimumab on the same day at a recommended doseof 3 mg/kg every 3 weeks for a total of 4 doses, and then nivolumab at arecommended dose of 240 mg every 2 weeks.

In certain embodiments, the optimized β-catenin nucleic acid inhibitormolecule is administered before the immunotherapeutic agent. In certainembodiments, the optimized β-catenin nucleic acid inhibitor molecule isadministered after the immunotherapeutic agent. In certain embodiments,the patient has been previously treated with the therapeutic agentbefore beginning treatment with the optimized β-catenin nucleic acidinhibitor molecule. The therapeutically effective amount of theoptimized β-catenin nucleic acid inhibitor molecule or immunotherapeuticagent may depend on the route of administration and the physicalcharacteristics of the patient, such as the size and weight of thesubject, the extent of the disease progression or penetration, the age,health, and sex of the subject and can be adjusted as necessarydepending on these and other factors.

EXAMPLES Example 1: Modified Beta-Catenin (BCAT) Nucleic Acid InhibitorMolecules

Several nucleic acid inhibitor molecules that target the beta-cateningene were made with different modification patterns to assess how themodification patterns might affect different properties of themolecules, such as immunogenicity, modulation of β-catenin mRNAexpression, and/or Ago2 binding. To this end, alternative structuralclasses of double-stranded nucleic acid inhibitor molecules were tested,including earlier generation, “non-extended” molecules having a bluntend at the right-hand side of the molecule (i.e., at 3′ terminus of thepassenger strand and 5′ terminus of the guide strand), as shown in FIG.1A, and next generation “extended” molecules having a guide strand witha single-stranded extension of 10 nucleotides at its 5′ terminus, asshown in FIG. 2A. In FIGS. 1A and 2A, unshaded nucleotide contain aribose, lightly shaded nucleotides contain a ribose with a 2′-OMemodification, the darker shaded nucleotides contain a ribose with a 2′-Fmodification, and nucleotides surrounded by a bolded box contain adeoxyribose.

Non-Extended Nucleic Acid Inhibitor Molecules

Three non-extended nucleic acid inhibitor molecules were constructed:NonExtend M1 (NEX M1), NonExtend M2 (NEX M2), and NonExtend M3 (NEX M3).NEX M1 has passenger and guide strands consisting of 25 and 27 basepairs, respectively. Together, the passenger and guide strands form aduplex region consisting of 25 base pairs with a two-base pair,single-stranded, overhang at the 3′ end of the guide strand. A number of2′-OMe modifications (lightly shaded boxes) were incorporated into thepassenger and guide strands of NEX M1. The last two nucleotides at the3′-terminus of the passenger strand contain a deoxyribose. FIG. 1A.

Several 2′-F modifications (darker shaded bases) were introduced intoNEX M1 to generate NEX M2. Except for the changes in the modificationpattern of the bases, the nucleotide sequences of the passenger andguide strands of NEX M2 are identical to those of NEX M1. FIG. 1A. Next,NonExtend M3 (NEX M3) was generated by adding four additional 2′-Fmodifications to NEX M2. The passenger and guide strands of NEX M2 andNEX M3 have the same nucleotide sequence, 25 base pair duplex region,and 3′ overhang as in NEX M1, the only differences being the 2′-Fmodification pattern.

Converting NEX M1 into an NEX M2 or NEX M3 structure did not affectintrinsic potency as demonstrated by the IC50 values of those constructsin cells (0.9 pM, 0.8 pM, and 0.8 pM, respectively), as shown in FIG.1A. To determine if the modification patterns in these sequences affectimmunogenicity of the double-stranded nucleic acid constructs, theinterferon response was measured after treating human PBMCs with NEX M1,NEX M2, and NEX M3. PBMCs were isolated from human blood samples fromdifferent donors by gradient centrifugation using Ficoll-Histophaque.Isolated PBMCs were treated with the double-stranded nucleic acidconstructs mixed with DOTAP (liposomal transfection reagent and immuneadjuvant) for different period of times and IF1T1 induction (aninterferon induced gene) was monitored by solid state cDNA synthesisfollowed by qPCR. An unmodified double-stranded nucleic acid inhibitormolecule (21 nucleotides in length) that targets the Bcl2 gene was mixedwith DOTAP (Avanti Polar Lipids, Alabaster, Ala.) and used as areference in this experiment. NEX M1 demonstrated significant elevationof IF1T1 while NEX M2 showed moderate elevation. NEX M3, on the otherhand, was very quiet compared to NEX M1 and NEX M2, suggesting that thespecific combination of 2′-OMe and 2′-F modifications in NEX M3substantially diminished the interferon response induced by NEX M3 inhuman PBMCs (FIG. 1B).

Extended Nucleic Acid Inhibitor Molecules

Next generation, extended, double-stranded nucleic acid constructs weregenerated by adding a 10-base pair single-stranded overhang at the5′-end of the guide strand. This 10-base pair single-stranded overhangalso includes additional 2′-OMe and 2′-F modifications. An extra C/Gbase pair was also incorporated at the right-hand side of the duplexbefore the 10-base pair single-stranded overhang, as shown in FIG. 2A.Incorporation of the extra C/G base pair and 10-base pair extension intothe NEX M1, NEX M2 and NEX M3 constructs produced the EX M1, EX M2 andEX M3 constructs, respectively, as shown in FIG. 2A.

Intrinsic potency of EX M1 was slightly affected but the incorporationdid not substantially affect the potency of EX M2 and EX M3 as shown bythe IC50 values in FIG. 2A. The interferon response of EX Ml, EX M2, andEX M3 in human PBMCs was measured as described above in Example 2. Atrend towards decreased immune stimulation was observed from EX M1 to EXM2 to EX M3 (FIG. 2B), similar to the trend observed in the nonextendedconstructs, suggesting that the specific combination of 2′-OMe and 2′-Fmodifications into the extended, double-stranded nucleic acid constructsreduces interferon responses.

Example 2: Further Evaluation of Selected BCAT Constructs

The nonextended construct with the smallest interferon response (NEX M3)was compared to the extended construct with the smallest interferonresponse (EX M3) for interferon response (as measured by IF1T1 inductionas previously described) in human PBMCs from 4 different donors. NEX M1was also included in the comparison.

NEX M3 and EX M3 showed essentially no interferon response in all 4donor PBMCs, whereas NEX M1 showed a modest interferon response whentreated with PMBCs from Donors 1, 2, and 4 and a substantial interferonresponse when treated with PMBCs from the hypersensitive Donor 3 (FIG.3A). Thus, the specific combination of 2′-OMe and 2′-F modifications inNEX M3 and EX M3 successfully decreased the interferon response causedby these nucleic acid constructs in human PBMCs.

Example 3: Beta-Catenin (CTNNBI) mRNA Knockdown in Tumors

To see how the modification patterns influence the target engagement intumors, the NEX M1, NEX M3, and EX M3 constructs were formulated inEnCore lipid nanoparticles (LNP) and tested for activity. The nucleicacid formulated LNPs were evaluated at different time points in tumorbearing mice. To generate tumors, 6-8 week old Hsd: AthymicNude-Foxn1^(nu) mice were injected subcutaneously with LS411N (5×10⁶cells) under the right shoulder. Tumor volume was measured every 2-3days to monitor tumor growth. When the tumors reached 200-250 mm³, theanimals were randomized and assigned to different cohorts and injectedwith LNPs carrying different double-stranded nucleic acid inhibitormolecule constructs as listed in FIG. 3B. LNP was administeredintravenously via lateral tail vein at a total volume of 10 ml/kg. Tumorsamples were collected at 24 and 120 hours after the dose and subjectedto mRNA analysis. EX M3 demonstrated slightly better potency whencompared to NEX M3 but had similar potency to NEX M1 24 hours afterdosing (FIG. 3B). However, EX M3 showed improved potency 120 hours afterdosing compared to NEX M1 but had similar potency to NEX M3. Based onthe interferon response and potency and duration effect, EX M3 wasselected for further characterization.

Example 4: Comparing NEX M1 to EX M3

The properties of EX M3 were further evaluated by comparing it with theearlier generation NEX M1. To investigate possible mechanisms for thisincreased potency, the exposure levels and levels of Ago2 binding (RISCincorporation) of each construct in the tumor homogenates were alsocompared. EX M3 (antisense strand) was detected in tumor tissue on day16 post tumor transplant at approximately 5-100× higher levels than NEXM1 (FIG. 4A) after treating the LS411N tumor bearing mice at 3 mg/kg for3 days (days 14, 15, and 16 post tumor transplant). In addition, on day16 post tumor transplant, EX M3 (antisense strand) showed approximately5-10× more Ago2 binding/RISC loading than NEX M1 (FIG. 4B) aftertreating the LS411N tumor bearing mice at 3 mg/kg for 3 days (days 14,15, and 16 post tumor transplant), demonstrating that EX M3 appears tobe more stable and active than the earlier generation NEX M1.

To see if the improved properties of EX M3 translated into anti-tumorefficacy, SW403 tumor bearing mice were treated with LNP-formulated NEXM1 or EX M3 along with Placebo and PBS at qdx3, 3 mg/kg dose levels (2cycles). After two weekly dosing cycles (qdx3, 3 mg/kg), NEX M1 inducedtumor growth inhibition of about 55% relative to vehicle-treatedanimals, whereas the EX M3 induced over 80% growth inhibition (FIG. 4C).At the end of the study, the tumors were also stained for beta-cateninprotein. As shown in FIG. 4D, there was a substantial decrease inbeta-catenin protein levels from the EX M3-treated tumors compared toNEX M1-treated tumors, suggesting that the improved potency and durationcaused by the EX M3 construct led to increased anti-tumor efficacy intumors treated with EX M3.

Example 5: Inhibiting β-Catenin in Wnt Active 4T1 Tumors

Balb/C mice were implanted with 4T1 tumors. At six days post 4T1 tumorcell implantation, with the average tumor size of 150-200 mm³, mice weresorted into two groups and were treated with either placebo or EX M3 at3 mg/kg on days 6 and 7 and days 12 and 13 post-implant, as shown inFIG. 7A. 48 hours after the last dose, tumors were collected and assayedby immunohistochemistry for β-catenin, CD8 and IDO1 protein levels. Asshown in FIG. 7B, EX M3 treatment decreased β-catenin levels andincreased CD8 levels but did not reduce the IDO1 levels significantlyafter two rounds of treatment.

In another study, 4T1 tumor cells were implanted in Balb/C mice and 4days post-implant, the mice were randomized into two groups and treatedwith placebo or EX M3. Mice were administered two doses of placebo or EXM3 at 3 mg/kg on days 4 and 5, as shown in FIG. 7C. This combinationdosing cycle was then repeated on days 9 and 10. Tumor growth wasmonitored by measuring the tumor sizes over the course of the treatmentperiod. Treating mice with EX M3 alone resulted in tumor growthinhibition of about 40%. FIG. 7C.

In another similar study, mice bearing 4T1 tumors were treated with PBSor EX M3 at 3 mg/kg on days 6 and 7 and days 12 and 13 post-implant, asshown in FIG. 8A. Tumors were collected 24 hours after the last dose andsubjected to flow cytometry to measure surface markers on single-cellsuspensions prepared from the extracted tumors. While the PBS controlhad no significant effect on the tumor immune microenvironment, EX M3treatment resulted in significant increases in cytotoxic T-cells (CD8),and multiple checkpoints (PD-1, LAG-3 and Tim-3). FIG. 8B. EX M3treatment significantly increased Regulatory T cells (Tregs), which playan important role in regulating or suppressing other cells of the immunesystem. FIG. 8B. No effect was observed on the immunosuppressive MDSCcells. FIG. 8B.

Example 6: Inhibiting IDO1 in Wnt Active 4T1 Tumors

Another efficacy study was performed in 4T1 tumors with the IDO1inhibitor, Epacadostat (IDOi). 4T1 tumor bearing mice were randomizedinto two groups and treated orally with vehicle or IDOi twice daily at100 mg/kg per dose on days 6 and 8 post-implant, as shown in FIG. 9A.Tumors were collected 48 hours after the last dose and were subjected toimmunohistochemistry to look at β-catenin, CD8 and IDO1 levels. IDOi at100 mg/kg reduced the IDO1 levels almost completely. FIG. 9B. β-cateninlevels were modestly decreased and CD8 levels were slightly increased.FIG. 9B. In a related study, mice bearing 4T1 tumors were administeredplacebo or IDOi twice daily at 100 mg/kg per day on days 6 and 8post-implant, as shown in FIG. 9C. Tumor growth was monitored bymeasuring the tumor sizes over the course of the treatment period.Treating mice with IDOi alone led to tumor growth inhibition, suggestingthat, in addition to β-catenin, the 4T1 tumors also depend on IDO1 fortumor growth. FIG. 9C.

Example 7: Inhibiting IDO1 in Wnt Active 4T1 Tumors in Combination withβ-Catenin Inhibition and/or a Checkpoint Inhibitor

Next, combination therapy in 4T1 tumors with EX M3 and IDOi or EX M3 anda checkpoint inhibitor (anti PD-1 antibody) or triple combinationtherapy with EX M3, IDO1, and an anti-PD-1 antibody was assessed. 4T1tumor bearing mice were sorted into 8 groups (n=5) and pre-treated twicedaily with IDOi (orally at 100 mg/kg per dose) on days 4 and 6post-implant and EX M3 or placebo (iv at 3 mg/kg per dose) on days 5 and6 post-implant, followed by anti-PD-1 antibody (ip at 5 mg/kg per dose)on days 7 and 8 post-implant, as shown in FIG. 10C. Mice also receivedEX M3, IDOi and PD-1 antibody as single agents (FIG. 10A) andcombinations of two agents (FIG. 10B). Mice receiving EX M3, IDOi, oranti-PD1 antibody as monotherapy showed modest anti-tumor efficacy. FIG.10A. The mice that received combination therapy with EX M3 and anti-PD-1antibody or EX M3 and IDOi demonstrated tumor stasis, reducing the rateof tumor growth. FIG. 10B. Remarkably, mice that were treated with allthree agents (EX M3, IDOi and anti-PD-1 antibody) demonstrated tumorregression, as shown in FIG. 10C, with pronounced reduction of the tumorvolume starting after administration of all three agents. Notably, asshown in FIG. 10C, the anti-tumor effect of the triple combination of EXM3, epacadostat (IDOi), and the anti-PD-1 antibody was markedly superiorto the effect observed with the double combination of epacadostat (IDOi)and the anti-PD-1, which is currently being evaluated in Phase IIIstudies.

At the end of the study (72 hours after the last anti-PD-1 antibodytreatment), tumors were collected and subjected to qPCR to analyzecertain T cell markers. There was a substantial increase in CD8 mRNAobserved in the mice that received the triple combination treatment ascompared to the other groups. FIG. 11A. FoxP3 is a marker forimmunosuppressive T cells called Tregs. Foxp3 mRNA levels were increasedwhen the anti-PD-1 antibody was added to either placebo or EX M3treatment. FIG. 11B. These levels were returned to background levelswith the addition of IDOi. FIG. 11B. Without intending to be bound byany theory, these mRNA data suggest that the triple combination of EXM3, IDOi, and anti-PD-1 antibody resulted in both a substantial increasein CD8 T cells and reduced levels of the immunosuppressive Tregs, andthat these changes in the T cell populations within the 4T1 tumormicroenvironment likely contributed to the observed tumor regression.

1.-20. (canceled)
 21. A method of treating a 0-catenin-associateddisorder in a subject, comprising administering to the subject atherapeutically effective amount of a nucleic acid inhibitor molecule incombination with one or more immunotherapeutic agents, wherein thef3-catenin-associated disorder is cancer, wherein the nucleic acidinhibitor molecule comprising a sense strand and an antisense strand anda region of complementarity between the sense strand and the antisensestrand of 26 nucleotides, wherein the sense strand comprises the nucleicacid of SEQ ID NO: 11; and wherein the antisense strand comprises thenucleic acid of SEQ ID NO: 12 and includes 2 single-stranded nucleotidesat its 3′ terminus and 10 single-stranded nucleotides at its 5′terminus.
 22. The method of claim 21, wherein the administeringcomprises intravenous, intramuscular, or subcutaneous administration.23. The method of claim 21, wherein the subject is a human.
 24. Themethod of claim 21, wherein the cancer is colorectal cancer,hepatocellular carcinoma, or melanoma.
 25. The method of claim 21,wherein the cancer is immunotherapy resistant.
 26. The method of claim21, wherein the immunotherapeutic agent is an antagonist of aninhibitory immune checkpoint molecule or an agonist of a co-stimulatorycheckpoint molecule.
 27. The method of claim 26, wherein theimmunotherapeutic agent is an IDO inhibitor, an anti-CTLA-4 monoclonalantibody, an anti-PD-1 monoclonal antibody, an anti-PD-L1 monoclonalantibody, or a combination of an anti-CTLA-4 monoclonal antibody and ananti-PD-1 monoclonal antibody.
 28. The method of claim 26, wherein theimmunotherapeutic agent is an IDO inhibitor.
 29. The method of claim 26,wherein the immunotherapeutic agent is an anti-PD-1 monoclonal antibody.30. The method of claim 26, wherein the immunotherapeutic agent is acombination of an IDO inhibitor and an anti-PD-1 monoclonal antibody.31. The method of claim 21, wherein the sense strand consists of thenucleic acid of SEQ ID NO:
 11. 32. The method of claim 21, wherein theantisense strand consists of the nucleic acid of SEQ ID NO:
 12. 33. Themethod of claim 21, wherein the sense strand consists of the nucleicacid of SEQ ID NO: 11 and the antisense strand consists of the nucleicacid of SEQ ID NO:
 12. 34. The method of claim 21, wherein the nucleicacid inhibitor molecule further comprising a 5′-phosphate mimic at the5′ terminus of the sense strand and/or the antisense strand.
 35. Themethod of claim 21, wherein the nucleic acid inhibitor molecule isformulated with a nanoparticle.
 36. The method of claim 35, wherein thelipid nanoparticle comprises core lipids and envelope lipids, whereinthe core lipids comprise a first cationic lipid and a first pegylatedlipid and wherein the envelope lipids comprise a second cationic lipid,a neutral lipid, a sterol, and a second pegylated lipid.
 37. The methodof claim 36, wherein the first cationic lipid is DL-048, the firstpegylated lipid is DSG-MPEG, the second cationic lipid is DL-103, theneutral lipid is DSPC, the sterol is cholesterol, and the secondpegylated lipid is DSPE-MPEG.
 38. A method of treating a0-catenin-associated cancer in a subject, comprising administering tothe subject a therapeutically effective amount of a nucleic acidinhibitor molecule in combination with an IDO inhibitor and an anti-PD-1monoclonal antibody; wherein the nucleic acid inhibitor moleculecomprising a sense strand and an antisense strand and a region ofcomplementarity between the sense strand and the antisense strand of 26nucleotides, wherein the sense strand comprises the nucleic acid of SEQID NO: 11; and wherein the antisense strand comprises the nucleic acidof SEQ ID NO: 12 and includes 2 single-stranded nucleotides at its 3′terminus and 10 single-stranded nucleotides at its 5′ terminus.
 39. Themethod of claim 38, wherein the nucleic acid inhibitor molecule furthercomprising a 5′-phosphate mimic at the 5′ terminus of the sense strandand/or the antisense strand.
 40. The method of claim 38, wherein thenucleic acid inhibitor molecule is formulated with a nanoparticle.